Many genes that predispose to human disease have now been identified and hundreds more will be found over the next decade. In order to take advantage of these findings, the biomedical community must be able to identify individuals that carry mutations in these genes in order to counsel them and to help them take preventative measures.
The methods now being used to screen for mutations in certain disease causing genes suffer from short comings that will make it far too time consuming and expensive to carry out large scale screenings. For example, the breast cancer susceptibility genes BRCA1 and BRCA2 recently have been identified, and mutations predisposing a human to breast cancer have been found throughout the 16 kilobases of coding sequence contained in these two genes. The entire coding sequences of BRCA1 and BRCA2 are now routinely screened by DNA sequencing, Single Strand Conformation Polymorphism (SSCP), or the Protein Truncation Test (PTT). All three of these techniques rely on the electrophoretic properties of either DNA or protein, and detection of the electrophoresed products is accomplished by incorporation of a labeled tag. The label may be either a fluorescent or radioactive. Sequencing both strands of the 16 kb of BRCA1/2 for a single individual is costly and tedious with current techniques and gives a large amount of redundant information, since virtually all of the sequence will be identical to the known sequence. SSCP is less tedious, but still requires sequencing benign polymorphisms. Furthermore, SSCP segment length is limited to 150 base pairs, requiring a minimum of 6 PCR reactions per kilobase screened. PTT is the fastest of these techniques but it is incapable of detecting missense mutations, and also suffers from all of the drawbacks of gel electrophoresis, such as gel preparation, long running times, and low resolution.
Other techniques such as Allele Specific Oligomer Hybridization and Allele Specific PCR are designed to identify one previously characterized mutation per assay. These techniques are useful when a particular mutation has already been identified and is widespread, but cannot be used for genes where a large number of yet unidentified mutations exist in the human population.
Thus, a need exists for a reliable, high throughput method for gene mutation detection which takes advantage of the speed, sensitivity, accuracy and resolving power of analytical measurement instruments such as mass spectrometry instruments.